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Original research article

Antibacterial Activity of Mahajak Remedy and Ingredients

Phadungnaree Saenmuang1, Sumalee Panthong 1,2, Arunporn Intharat1,2,*

1Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University, Pathum Thani 12120, Thailand 2Center of Excellence in Applied Thai Traditional Medicine Research, Faculty of Medicine, Thammasat University, Pathum Thani 12120, Thailand

Received 28 October 2019; Received in revised form 20 June 2020 Accepted 26 June 2020; Available online 21 September 2020

ABSTRACT Mahajak (MH) oil is a remedy in Tamra-Osodpranarai. It is used for relieving pain, and skin infection as a Thai traditional medicine. This study investigated the efficacy of the MH remedy and plant ingredient extract on antibacterial activities. The MH remedy was macerated with oil, 95% ethanol, hexane, and its plant ingredients were macerated with 95% ethanol. Then, all extracts were concentrated by rotary evaporator. The extracts were investigated on antibacterial activity against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 9027, and Bacillus subtilis ATCC 6633 by disc diffusion method, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The results show that the 95% ethanol extract of MH remedy was the best extract of antibacterial testing. Using the disc containing 10 µL (concentrate 500 mg/mL of the extracts), the inhibition zone was observed against S. aureus (9.67±0.33 mm). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 2.50 mg/mL were obtained for residue hexane. Hexane extracts showed MIC and MBC of S.aureus and B.subtilis with similar values to 95% ethanol. However, all of the tests had no inhibitory activity against P.aeruginosa. In conclusion, this study demonstrated that 95% ethanol extracts of the MH remedy have antibacterial activity and may be a potential treatment in cases of skin infection.

Keywords: Antibacterial activities; Mahajak oil; Tamra-osodpranarai

*Corresponding author: [email protected] doi: 10.14456/scitechasia.2020.37 P. Saenmuang et al. | Science & Technology Asia | Vol.25 No.3 July – September 2020 1. Introduction However, there is no scientific report Bacterial skin infections are caused by on the antibacterial activity of this remedy. many types of bacteria such as Thus, the objective of this study was to Staphylococcus aureus, Pseudomonas investigate the antibacterial activity of MH aeruginosa, and Bacillus subtilis [1]. Skin extracts involved in skin infection and infection includes cellulitis, erysipelas, inflammation. impetigo, folliculitis, furuncles and carbuncles, which can be classified by the 2. Materials and methods depth and skin compartment [2, 3]. The 2.1 Plant materials World Health Organization (WHO) has The MH remedy, including Nigella estimated that 56.9 million people died sativa L, Lepidium sativum L, Cuminum worldwide in 2016, and infectious diseases cyminum L, Foeniculum vulgare Miller, were one cause of death [4]. Antibiotics, such Anethum graveolens L., and Piper as penicillin, clindamycin, and retrofractum Vahl., was obtained from a Thai cephalosporin, are widely used to treat skin herbal drug store in Bangkok. The infection [5]. However, bacteria that still ingredients were confirmed by comparison survive on antibiotic treatment are called with authentic voucher specimens that were antibiotic-resistant. Antibiotic resistance has kept in the herbarium of Southern Center of become a global health problem. In 2018, the Thai Medicinal , Faculty of Phar- National Antimicrobial Resistance maceutical Sciences, Prince of Songkla Surveillance Center in Thailand reported that University, Songkhla, Thailand. The herbar- antibiotic resistance was found in all parts of ium voucher specimen number is shown in Thailand, such as S. aureus resistant to (Table 1). Plant ingredients were washed methicillin [6]. At the present time, many with water to remove contamination and researchers are interested in an antimicro- dried at 50ºC for 72 hr. Each plant was bial agent from a natural product. Secondary ground to powder and weighed according to metabolites from plants, such as tannins, the recipe of the MH remedy in the alkaloids, and quinones, can inhibit patho- Osodpranarai scripture. gens. Thus, many natural products have been investigated as antimicrobial agents and in 2.2 Preparation of crude extract the development of antimicrobial agents [7]. Plant ingredients of the MH remedy In Thailand, traditional Thai doctors have were extracted by the traditional method and used Thai traditional remedies that combine conventional method. For the traditional many types of herbal plant as a drug for the method, plant ingredients were macerated in treatment of patients for a long time. sesame oil, while an organic solvent such as Mahajak (MH) is a traditional Thai remedy ethanol and hexane were used for the which has been described in the conventional extraction method. Osodphranarai scripture. It has long been 2.2.1 Maceration in sesame oil used to relieve pain and reduce skin Firstly, fresh peel of kaffir lime was inflammation. At present, the MH remedy is added into sesame oil (the ratio of 2:1) and still used by Thai Traditional doctors in heated at 150ºC for 25-30 min. Then, the Lopburi’s hospital to treat skin inflame- sesame oil was filtered, cooled down to room mation. The MH remedy consists of Nigella temperature, and used for maceration of plant sativa L., Lepidium sativum L., Cuminum ingredients. Plant powder ingredients were cyminum L., Foeniculum vulgare Miller., separated into two parts. The first part and Anethum graveolens L., Piper retrofractum second part of plant ingredients were Vahl., Citrus hystrix DC., and Sesamum macerated with a sesame oil volume of 600 indicum L. [8,9]. mL and 200 mL for 15 days to obtain MH oil

79 P. Saenmuang et al. | Science & Technology Asia | Vol.25 No.3 July – September 2020 remedy extract (MH1) and MH oil 2.3.1 Loss on drying concentrate extract (MH2). The electronic moisture analyzer 2.2.2 Maceration in an organic was used for the analysis of loss on drying. solvent Each plant (2 g) was put into the moisture The crude powder was extracted by analyzer and heated to 105°C. The weight maceration with hexane and 95% ethanol for (W) of the dried sample was taken and three days and filtered through Whatman analyzed by using the following equation. filter paper no1. The maceration process was W start (g) − W dried repeated twice more with hexane and 95% %Loss on drying = × 100 ethanol. The hexane extract (MH3) and W start (g) ethanolic extract (MH4) were concentrated by rotary evaporator, vacuum dried for one 2.3.2 Ethanol-soluble extractive day, and stored at -20ºC until used. The value residues of MH1, MH2, and MH3 were Five grams of the powdered drug extracted by maceration with 95% ethanol was macerated with 100 mL of ethanol in a and used the same procedure as MH4 flask that was closed with foil for 24 hrs. extraction. Finally, we obtained MH (Oil Moreover, it was occasionally shaken over remedy) extract (MH5), residue of MH (Oil the first 6 hrs. Then, it was allowed to stand concentrate) extract (MH6), the residue of for 18 hrs. and filtered. After filtration, 25 hexane extract (MH7). mL of the filtrate was evaporated at 105°C to The yields of MH1- MH7 extracts dryness in a tare flat-bottomed shallow dish, were calculated as the percentage of yield by then dried and weighed. Percentage of using the following equation: ethanol-soluble extractive value was calculated with the below formula Weight of dried extract (g) %Yield = × 100 W extract (g) Weight of dried crude power (g) %Extractive value = × 100 W dried plant material (g) 2.3 Quality control of plant ingredients [10] 2.3.3 Total ash All plant ingredients were tested for This method was used to investigate quality control including loss on drying, the physiological ash and non-physiological extractive value, total ash, and insoluble acid ash or inorganic compounds of plant ash by using the guidelines of the Thai material. Herbal Pharmacopoeia.

Table 1. Plant materials of MH remedy.

Voucher Part Weight Scientific Name Family Name Thai Name specimen formulation number L. Ranunculaceae Thian-Dam Seed SKP 160 14 19 01 15 g Lepidium sativum L. Cruciferae Thian-Daeng Seed SKP 057 12 19 01 15 g Cuminum cyminum L. Umbelliferae Thian-Khao Seed SKP 199 03 03 01 15 g Foeniculum vulgare Miller subsp. Umbelliferae Thian- Khaoplueak Seed SKP 199 06 22 01 15 g Anethum graveolens L. Umbelliferae Thian- Tatakkataen Seed SKP 199 01 07 01 15 g

Piper retrofractum Vahl. Dipli Fruit SKP 146 16 18 01 30 g

80 P. Saenmuang et al. | Science & Technology Asia | Vol.25 No.3 July – September 2020 Total ash was determined by bacterium was swabbed on Mueller Hinton weighing 2 g into a pre-weighed crucible and agar (MHA). Air-dried discs were placed on burning in a muffle furnace at 450°C for 5 hr. the inoculated MHA surface. An antibiotic Then, the crucible was placed into the commercial disc of ampicillin 10 µg/disc was desiccator to cool down. Finally, crucible used as a positive control. Negative control with ash was weighed again. The percentage was a disc that contained 10 µL of 2% of total ash was calculated by using the DMSO. Plates were incubated at 37ºC for 18- following equation. 24 hr. After incubation, the inhibition zone diameter was measured. These tests were W after burning (g) %Total ash = × 100 performed in triplicate. W before burning (g) 2.4.2 Determination of minimum inhibitory concentration (MIC) [12] 2.3.4 Acid insoluble ash The MIC value was determined by a The ash obtained from the previous broth dilution method. Briefly, all extracts process was boiled with 25 mL of 10 % HCl were dissolved in 2% dimethylsulfoxide for 5 min. The insoluble matter was collected (DMSO) in which the maximum final on ash-less filter paper and washed with DI concentration was 5 mg/mL. The serial two- water (pH 7). Ash-less filter paper with the fold dilution was performed in a insoluble matter was dried and weight. After concentration range of 0.039-5.00 mg/mL in that, it was burned and cooled in a desiccator. a 96-well plate (50 µL/well). Then, 50 µL of Finally, it was weighed and the percentage of 0.5 McFarland of the tested microorganisms acid insoluble ash was calculated by using was added to each well and incubated at 37°C the following equation. for 24 hr. After that, 10 µl of resazurin (5 mg/mL) was added into each well and W after burning (g) %Acid insoluble ash = × 100 incubated at 37°C for 2 hr. The final W before burning (g) concentration that did not show a color change of resazurin from blue to pink was the 2.4 Anti-bacterial Assay MIC value. The microorganisms used in this 2.4.3 Determination of minimum study were obtained from the Faculty of bactericidal concentration (MBC) [13] Medicine, Thammasat University, Khlong The MBC value is the lowest extract Luang, Pathum Thani, Thailand. The refer- concentration of extract which can kill ence bacterial strains were Staphylococcus bacteria at least 99.9% within 24 hr. In short, aureus ATCC 25923, Pseudomonas aeru- the contents in the MIC well and those above ginosa ATCC 9027, and Bacillus subtilis the MIC for each micro-organism was ATCC 6633. All of the bacteria were streaked on MHA and incubated at 37°C for cultured on nutrient agar and incubated for 24 hr. After incubation, the bacteria colonies 24 hr. at 37ºC before testing. on MHA were observed. The final 2.4.1 Antibacterial assay by Disc concentration that bacteria colony did not diffusion method [11] appear was the MBC value. The disc diffusion was used to screen the antibacterial activity of extracts. All 3. Results and Discussion extracts were prepared at 500 mg/mL in MH1 showed the highest yield with DMSO and transferred (10 µL) to paper disc. a percent yield value of 61.17%. Moreover, Each bacterium was inoculated in Mueller MH2 and MH7 showed a high percent yield Hinton broth (MHB) and incubated at 37ºC with a percentage of yield of 40.68% and for 2 hr. After that, it was adjusted to 0.5 35.67%, respectively, as shown in Table 2. McFarland by using a McFarland TD1 showed the highest yield (32.80%) densitometer. Then, 0.5 McFarland of each 81 P. Saenmuang et al. | Science & Technology Asia | Vol.25 No.3 July – September 2020 when compared with other plant ingredients. of sand, soil, and siliceous earth in plant Moreover, TK1 and DP also showed a high ingredients, total ash and insoluble acid ash percent yield with 10.99% and 10.47%. of plant were performed. All plant materials Quality control of plant ingredients displayed the total ash value of percent total of the Mahajak remedy was performed ash between 0.43-7.15% and the insoluble including extractive value testing, loss on acid ash of percent acid insoluble ash drying testing, total ash testing, and acid between 0.62-1.76%. insoluble ash testing, as shown in Table 3. The MH remedy extracts and its The results of extractive value showed that plant ingredients extracts were investigated TD2, TD1, and TK3 showed high extractive for antibacterial activity by using the disk

Table 2. The percentage yield of MH extracts and its plant ingredients.

Sample Code % Yield Mahajak (Oil remedy) extract MH1 61.17 Mahajak (Oil concentrate) extract MH2 40.68 Hexane extract MH3 21.09 95% Ethanol extract MH4 25.11 The residue of MH (Oil remedy )extract MH5 27.77 Residue of MH (Oil concentrate) extract MH6 20.43 Residue of Hexane extract MH7 35.67 Nigella sativa L. TD1 32.80 Lepidium sativum L. TD2 8.01 Cuminum cyminum L. TK1 10.99 Foeniculum vulgare Miller subsp. TK2 8.29 Anethum graveolens L. TK3 6.07 Piper retrofractum Vahl. DP 10.47

Table 3. Quality control of MH remedy extract and plant ingredients.

Loss on drying % Total Ash % Total Acid % Extractive Plant Criteria % Loss on Standard Standard value standard drying <10% <2% Nigella sativa L. 14.67± 0.47 7 % 5.72 ± 0.37 0.43 ± 0.02 0.62 ± 0.19 Lepidium sativum L. 23.69 ± 0.36 10 % 6.72 ± 0.09 5.10 ± 0.02 0.91 ± 0.14 Cuminum cyminum L. 6.10 ± 0.32 10 % 9.21 ± 0.08 5.57 ± 0.02 1.44 ± 0.10 Foeniculum vulgare Miller subsp. 10.04 ± 0.25 10 % 7.52 ± 0.22 7.15± 0.11 1.34 ± 0.18 Anethum graveolens L. 11.50 ± 0.12 9 % 7.44 ± 0.28 5.48 ± 0.08 0.69 ± 0.12 Piper retrofractum Vahl. 3.56 ± 0.02 13 % 9.63 ± 0.28 6.45 ± 0.08 1.25 ± 0.10 MH remedy 6.50 ± 0.05 10 % 3.76 ± 0.37 6.04 ± 0.26 1.76 ± 0.07 values of 23.69%, 14.67%, and 11.50%, diffusion method. All extracts have no respectively. For loss on drying, we found inhibitory effect on P. aeruginosa. MH4 that all samples passed the standard criteria showed the best activity to inhibit S. aureus of the Thai Herbal Pharmacopeia. and B. subtilis with an inhibition zone of 9.67 The results are shown in quality and 10.33 mm, respectively, as shown in control of the MH remedy and plant Table 4. ingredients. To determine the contamination

82 P. Saenmuang et al. | Science & Technology Asia | Vol.25 No.3 July – September 2020 Moreover, MH3 and MH7 showed The antibacterial activity of plant antibacterial activity against S. aureus with ingredient extract of MH remedy is presented an inhibition zone of 9.33 and 8.33 mm, in Table 6. All extracts have no inhibitory respectively. However, some MH extract effect on P. aeruginosa and S. aureus. showed no effect against S. aureus and B. However, the TD1 extract showed the best subtilis such as MH1, MH2, MH5, and MH6. activity to inhibit B. subtilis, with an Table 4. Antibacterial activities of MH inhibition zone of 19.50 mm, followed by remedy extracts against bacterial test TK2 with the inhibition zone of 17.00 mm. Moreover, TD2 and TK1 have also an organisms. inhibitory effect on B. subtilis, which has the MH Zone of inhibition in mm. extracts S. aureus P. aeruginosa B. subtilis inhibitory area equal to 10.40 and 8.00 mm, MH1 NI NI NI respectively, as shown in Table 6. MH2 NI NI NI MH3 NI NI 9.33±0.33 MH4 9.67±0.33 NI 10.33±0.67 MH5 NI NI NI MH6 NI NI NI MH7 NI NI 8.33±0.33 AM 28.33±0.17 NI 30.13±0.09

All extracts were tested for antibacterial activity by using a microtiter plate-based method to confirm their antibacterial activity. The MH4 extract showed the best antibacterial activity against S. aureus and B. subtilis with MIC values of 2.50 and 1.25 mg/mL and MBC values of

2.50 and 2.50 mg/mL, respectively. MH3 and MH7 showed an inhibition zone against B. subtilis that displayed MIC values of 2.50 and 5.00 mg/mL, respectively. However, MH3 and MH7 showed no killing effect on S. aureus. Furthermore, all extracts showed no effect against P. aeruginosa, as shown in Table 5.

Table 5. The MIC (mg/mL) and MBC (mg/mL) of the MH remedy extract.

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Table 7. The MIC (mg/mL) and MBC (mg/mL) of plant ingredient extract of MH remedy. The value of minimum inhibitory concentration and plant ingredient minimum bactericidal concentration extract S. aureus P. aeruginosa B. subtilis MIC MBC MIC MBC MIC MBC TD1 >5 >5 >5 >5 0.5 0.5 TD2 >5 >5 >5 >5 0.5 0.5 TK1 0.5 0.5 >5 >5 0.625 0.625 TK2 0.5 0.5 >5 >5 0.25 0.25 TK3 >5 >5 >5 >5 >5 >5 DP >5 >5 >5 >5 >5 >5 AM 0.195 0.195 2.5 2.5 0.0976 0.0976

From Table 7, it was found that the [15]. can be dissolved in the organic extract from TK2 shows the best antibacterial solvent [16], and we also found that F. activity against B. subtilis with MIC and vulgare had an antibacterial activity against MBC at 0.25 mg/mL. TD1 and TD2 show S. aureus and P. aeruginosa following the antibacterial activity against B. subtilis at activity against B. subtilis. Many plant 0.50 mg/mL, while TK1 and TK2 resisted S. components of the MH remedy contain an aureus bacteria by showing MIC and MBC essential oil that can increase the values at 0.50 mg/mL which is considered permeability of bacterial cell membranes and acceptable. From the tests of all extracts, induce bacterial cell death [17]. Moreover, there is no extract against P. aeruginosa. plant components of the MH remedy, such as The Mahajak remedy that is N. sativa oil, can inhibit multi-drug resistant mentioned in the Tamra-Osodphranarai is S. aureus that was isolated from a diabetic’s used to relieve pain and skin infection after patient wound and can increase the patient’s injury [8]. Therefore, this research wound healing [18]. The N. sativa extract investigated the antibacterial activity of MH was nearly as effective as the standard drug, remedy extract with various extractions by mupirocin, and showed no side effect [19]. taking all the components of Mahajak When compared with the experiments done, remedy to test. The results show that gram- it was found that TD1 could inhibit some positive bacteria were more sensitive to the skin-related bacteria. Previous reports about MH extract than gram-negative bacteria. The P. retrofractum (DP) showed that the 95% ethanolic extract of MH remedy (MH4) methanolic extract of P. retrofractum extract showed the highest antibacterial activity inhibits S. aureus and B. subtililis with MIC against S. aureus and B. subtilis while the and MBC values of 225 µg/mL [20]. The hexane extract and the 95% ethanolic extract results of the experiment may be due to DP of hexane residue have little effect against contains alkaloid compounds such as results B. subtilis. of the experiments in this work that found the Moreover, the MH remedy value of TK2 shows the best antibacterial contained P. retrofractum as a major activity with B. subtilis with MIC and MBC ingredient. Thus, the hexane extract of MH at 0.25 mg/mL [21]. There was a research remedy may contain non-polar chemical report on Lepidium sativum L. (TD2) seed oil substances from P. retrofractum such as that revealed anti-inflammatory and piperine and piperanine [14]. Piperine can antimicrobial activity against S. aureus, inhibit S. aureus (50 µg/mL) and increase the B. subtilis, P. aeruginosa [22]. The ability of ciprofloxacin to reduce S. aureus antimicrobial activity of essential oil from

84 P. Saenmuang et al. | Science & Technology Asia | Vol.25 No.3 July – September 2020 (Anethum graveolens L.) from seeds Thammasat University, for financial support investigated in terms of the disc-diffusion of this research. method showed the best antimicrobial activity against S.aureus [23]. Moreover, the References essential oils from the fruit peel of Citrus [1] Daniel L, Marc A, Richard A. Common hystrix DC have antibacterial activity [24]. In Bacterial Skin Infections. Am Fam this research, the residue of hexane extract is Physician. 2002;66(1):119-25. extracted by maceration with 95% ethanol to obtain MH4 extract. It also showed [2] Stulberg DL, Penrod MA, Blatny RA. antibacterial activity against B. subtilis Common bacterial skin infections. Am Fam Physician 2002;66(1):119-24. because it may contain polar substances that are extracted by ethanol. All plants in the MH [3] Sukumaran V, Senanayake S. Bacterial remedy contain similar components, such as skin and soft tissue infections. Australian volatile oil, flavonoids, alkaloids, and prescriber 2016;39(5):159-63. coumarins. These chemical compounds also show antibacterial and anti-inflammatory [4] Hogberg L, Heddini A, Cars O. The global activity and reduce muscle pain [25]. need for effective antibiotics: challenges However, MH4 showed lower antibacterial and recent advances. Trends Pharmacol activity than its plant ingredients, which may Sci 2010;31(11):509-15. be caused by the proportion of each plant [5] Sunderkotter C, Becker K. Frequent ingredient in MH4 being 14-28%. Thus, bacterial skin and soft tissue infections: MH4 showed low antibacterial activity. diagnostic signs and treatment. JDDG. Moreover, all of the extracts, including MH 2015;13(6):501-24. and plant ingredients, also showed low antibacterial activity when compared with [6] National Antimicrobial Resistance ampicillin as a positive control. Ampicillin is Surveillance Center Thailand (NARST) . a pure compound that inhibits pathogens Antimicrobial Resistance Surveillance [26]. While MH extract and plant ingredient Center Thailand. Antimicrobial extracts contained many active compounds Resistance 2000-2018 [Internet]. [cited that may inhibit bacteria. Active compounds 2019 Jul 17]. Available from: http://narst.dmsc.moph.go.th/ in MH may show good effect on antibacterial activity when it is isolated from plant extracts [7] Brown DG, Lister T, May-Dracka TL. and MH remedy extracts. New natural products as new leads for antibacterial drug discovery. Bioorg Med 4. Conclusion Chem Lett. 2014;24(2):413-8. In conclusion, our results provided evidence that the MH remedy extract has [8] Thai and Alternative Medicine Division. moderate antibacterial activity. Further- King Narai Hospital. Tamra PhraOsot Phranarai [Brochure]; Lopburi; 2016. more, chemical constituents of the Mahajak remedy and biological activities that involve [9] Phichiansunthon C, Chawalit M, wound healing and pain relief should be Chairawong W. Tamra PhraOsot performed in the future. Phranarai. 1st ed. Bangkok: Amrin and Phoompanya Foundation; 2001. Acknowledgements We would like to thank the Center of [10] Ministry of Public Health. Thai herbal Excellence in Applied Thai Traditional pharmacopoeia. Vol. Supplement. Medicine Research, Faculty of Medicine, Nonthaburi: Department of Medical Sciences, Ministry of Public Health; 2004.

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